PeerJ Preprints: Histologyhttps://peerj.com/preprints/index.atom?journal=peerj&subject=1760Histology articles published in PeerJ PreprintsThe anatomy of the foveola reinvestigatedhttps://peerj.com/preprints/265182018-02-132018-02-13Alexander V. TschulakowTheo OltrupThomas BendeSebastian SchmelzleUlrich Schraermeyer
Objective. In the foveola of the eye, photoreceptors and Müller cells with a unique morphology have been described, but little is known about their 3D structure and orientation. Considering that there is an angle-dependent change in the foveolar photoreceptor response for the same light beam, known as the Stiles Crawford Effect of the first kind (SCE I), which is still not fully understood, a detailed analysis of the anatomy of the foveolar cells might help to clarify this phenomenon. Methods. Serial semithin and ultrathin sections, and focused ion beam (FIB) tomography were -prepared from 32 foveolae from monkeys (Macaca fascicularis) and humans. Foveolae were also analyzed under the electron microscope. Serial sections and FIB analysis were then used to construct 3D models of central Müller and photoreceptor cells. In addition, we measured the transmission of collimated light under the light microscope at different angles after it had passed through human foveae from flat mounted isolated retinae. Results. In monkeys, outer segments of central foveolar cones are twice as long as those from parafoveal cones and do not run completely parallel to the incident light. Unique Müller cells are present in the central foveolae (area of 200 µm in diameter) of humans and monkeys. Light entering the fovea center, which is composed only of cones and Müller cells, at an angle of 0 degrees causes a very bright spot after passing through this area. However, when the angle of the light beam is changed to 10 degrees, less light is measured after transpasssing through the retina, the foveolar center becomes darker and the SCE-like phenomenon is directly visible. Measurements of the intensities of light transmission through the central foveola for the incident angles 0 and 10 degrees resemble the relative luminance efficiency for narrow light bundles as a function of the location where the beam enters the pupil as reported by Stiles and Crawford. The effect persisted after carefully brushing away the outer segments. Conclusion. We show that unique cones and Müller cells with light fibre-like properties are present in the center of the fovea. These unique Müller cells cause an angle dependent, SCE-like drop in the intensity of light guided through the foveola. Outer segments from the foveola cones of monkeys are not straight.

Objective. In the foveola of the eye, photoreceptors and Müller cells with a unique morphology have been described, but little is known about their 3D structure and orientation. Considering that there is an angle-dependent change in the foveolar photoreceptor response for the same light beam, known as the Stiles Crawford Effect of the first kind (SCE I), which is still not fully understood, a detailed analysis of the anatomy of the foveolar cells might help to clarify this phenomenon. Methods. Serial semithin and ultrathin sections, and focused ion beam (FIB) tomography were -prepared from 32 foveolae from monkeys (Macaca fascicularis) and humans. Foveolae were also analyzed under the electron microscope. Serial sections and FIB analysis were then used to construct 3D models of central Müller and photoreceptor cells. In addition, we measured the transmission of collimated light under the light microscope at different angles after it had passed through human foveae from flat mounted isolated retinae. Results. In monkeys, outer segments of central foveolar cones are twice as long as those from parafoveal cones and do not run completely parallel to the incident light. Unique Müller cells are present in the central foveolae (area of 200 µm in diameter) of humans and monkeys. Light entering the fovea center, which is composed only of cones and Müller cells, at an angle of 0 degrees causes a very bright spot after passing through this area. However, when the angle of the light beam is changed to 10 degrees, less light is measured after transpasssing through the retina, the foveolar center becomes darker and the SCE-like phenomenon is directly visible. Measurements of the intensities of light transmission through the central foveola for the incident angles 0 and 10 degrees resemble the relative luminance efficiency for narrow light bundles as a function of the location where the beam enters the pupil as reported by Stiles and Crawford. The effect persisted after carefully brushing away the outer segments. Conclusion. We show that unique cones and Müller cells with light fibre-like properties are present in the center of the fovea. These unique Müller cells cause an angle dependent, SCE-like drop in the intensity of light guided through the foveola. Outer segments from the foveola cones of monkeys are not straight.

Morphohistological development of the somatic embryo of Typha domingensishttps://peerj.com/preprints/264592018-01-232018-01-23Guadalupe Hernández-PiedraVioleta Ruiz-CarreraAlberto J SánchezArlette Hernández-FranyuttiAlfonso Azpeitia-Morales
Background. The sustainable methods of propagation for Typha domingensis through somatic embryogenesis can help to mitigate its current condition of ecological marginalisation and overexploitation. Then, the hypothesis established that the variation of the concentration of auxin and light conditions in sequential stages of culture generate different morphogenetic routes that can be monitoring by morphohistological markers. Methods. Murashige and Skoog medium at half ionic strength, 3% sucrose and 0.1% ascorbic acid were used in the induction, proliferation and embryogenic maturation. Induction started with aseptic germinates cultured in 0.5 mg L-1 of 2,4-dichlorophenoxyacetic. Four concentrations of 0 to 2 mg L-1 of 2,4-dichlorophenoxyacetic, that generated four embryogenic lines, were evaluated in darkness. Maturation of the somatic embryo took place, in each embryogenic line, without auxin and under light and dark conditions. Results. The yellow and brown callus, as well as oblong and scutellar somatic embryos were recorded in the methodological sequence. The embryogenic differentiation was described with histological analysis. The induced cultures produced both somatic embryos in a small proportion. The percentages of the yellow callus on the explant and of suspended cells in the embryogenic proliferation were greater with the three concentrations of 2,4-dichlorophenoxyacetic. While, the brown callus predominated without auxin. The somatic embryo developed under light and dark conditions, and presented globular, oblong, scutellar and sparsely coleoptilar stages. Discussion. The combined effect of auxin concentrations and light-dark conditions generated conditions that favoured the development of embryogenic calluses and somatic embryos (globular, oblong, scutellar and coleoptilar) in an asynchronous process with respect to the stages of embryogenic induction, proliferation, and maturation. Indeed, differentiation and cellular organization of this process were compatible with descriptors of the embryogenic stages recorded by other aquatic and terrestrial monocotyledons.

Background. The sustainable methods of propagation for Typha domingensis through somatic embryogenesis can help to mitigate its current condition of ecological marginalisation and overexploitation. Then, the hypothesis established that the variation of the concentration of auxin and light conditions in sequential stages of culture generate different morphogenetic routes that can be monitoring by morphohistological markers. Methods. Murashige and Skoog medium at half ionic strength, 3% sucrose and 0.1% ascorbic acid were used in the induction, proliferation and embryogenic maturation. Induction started with aseptic germinates cultured in 0.5 mg L-1 of 2,4-dichlorophenoxyacetic. Four concentrations of 0 to 2 mg L-1 of 2,4-dichlorophenoxyacetic, that generated four embryogenic lines, were evaluated in darkness. Maturation of the somatic embryo took place, in each embryogenic line, without auxin and under light and dark conditions. Results. The yellow and brown callus, as well as oblong and scutellar somatic embryos were recorded in the methodological sequence. The embryogenic differentiation was described with histological analysis. The induced cultures produced both somatic embryos in a small proportion. The percentages of the yellow callus on the explant and of suspended cells in the embryogenic proliferation were greater with the three concentrations of 2,4-dichlorophenoxyacetic. While, the brown callus predominated without auxin. The somatic embryo developed under light and dark conditions, and presented globular, oblong, scutellar and sparsely coleoptilar stages. Discussion. The combined effect of auxin concentrations and light-dark conditions generated conditions that favoured the development of embryogenic calluses and somatic embryos (globular, oblong, scutellar and coleoptilar) in an asynchronous process with respect to the stages of embryogenic induction, proliferation, and maturation. Indeed, differentiation and cellular organization of this process were compatible with descriptors of the embryogenic stages recorded by other aquatic and terrestrial monocotyledons.

­Postcranial skeletal anatomy of the holotype and referred specimens of Buitreraptor gonzalezorum Makovicky, Apesteguía and Agnolín 2005 (Theropoda, Dromaeosauridae), from the Late Cretaceous of Patagoniahttps://peerj.com/preprints/264502018-01-222018-01-22Federico A GianechiniPeter J MakovickySebastián ApesteguíaIgnacio Cerda
Here we provide a detailed description of the postcranial skeleton of the holotype and referred specimens of Buitreraptor gonzalezorum. This taxon was recovered as a unenlagiine dromaeosaurid in several recent phylogenetic studies and is the best represented Gondwanan dromaeosaurid discovered to date. It was preliminarily described in a brief article, but a detailed account of its osteology is becoming known for recent work. Hitherto, the holotype is the most complete specimen found, so its exhaustive description provides much valuable anatomical information. The holotype and referred specimens preserve the axial skeleton, pectoral and pelvic girdles, and both fore- and hindlimbs. Diagnostic postcranial characters of this taxon include: anterior cervical centra exceeding the posterior limit of neural arch; antepenultimate and penultimate cervical vertebral centra with lateroventral tubercles; pneumatic foramina only in anteriormost dorsals; medial and distal caudal centra with a complex of ridges on lateral surfaces; pneumatic furcula with two pneumatic foramina on the ventral surface; scapular blade transversely expanded at mid-length; well-projected flexor process on distal humerus; dorsal rim of the ilium laterally everted; and concave dorsal rim of the postacetabular iliac blade. A paleohistological study of limb bones shows that the holotype represents a younger ontogenetic stage than one of the referred specimens (MPCA 238), which correlates with the fusion of the last sacral vertebra to the rest of the sacrum in MPCA 238. A revised phylogenetic analysis recovered Buitreraptor as a unenlagiine dromaeosaurid, in agreement with previous works. The phylogenetic implications of the unenlagiine synapomorphies and other characters, such as the specialized pedal digit II and the distal ginglymus on metatarsal II, are discussed within the evolutionary framework of Paraves.

Here we provide a detailed description of the postcranial skeleton of the holotype and referred specimens of Buitreraptor gonzalezorum. This taxon was recovered as a unenlagiine dromaeosaurid in several recent phylogenetic studies and is the best represented Gondwanan dromaeosaurid discovered to date. It was preliminarily described in a brief article, but a detailed account of its osteology is becoming known for recent work. Hitherto, the holotype is the most complete specimen found, so its exhaustive description provides much valuable anatomical information. The holotype and referred specimens preserve the axial skeleton, pectoral and pelvic girdles, and both fore- and hindlimbs. Diagnostic postcranial characters of this taxon include: anterior cervical centra exceeding the posterior limit of neural arch; antepenultimate and penultimate cervical vertebral centra with lateroventral tubercles; pneumatic foramina only in anteriormost dorsals; medial and distal caudal centra with a complex of ridges on lateral surfaces; pneumatic furcula with two pneumatic foramina on the ventral surface; scapular blade transversely expanded at mid-length; well-projected flexor process on distal humerus; dorsal rim of the ilium laterally everted; and concave dorsal rim of the postacetabular iliac blade. A paleohistological study of limb bones shows that the holotype represents a younger ontogenetic stage than one of the referred specimens (MPCA 238), which correlates with the fusion of the last sacral vertebra to the rest of the sacrum in MPCA 238. A revised phylogenetic analysis recovered Buitreraptor as a unenlagiine dromaeosaurid, in agreement with previous works. The phylogenetic implications of the unenlagiine synapomorphies and other characters, such as the specialized pedal digit II and the distal ginglymus on metatarsal II, are discussed within the evolutionary framework of Paraves.

Ampelisca eschrichtii Krøyer, 1842 (Ampeliscidae) of the Sakhalin Shelf in the Okhotsk Sea starve in summer and feast in winterhttps://peerj.com/preprints/34962017-12-282017-12-28Valentina B. DurkinaJohn W. ChapmanNatalia L. Demchenko
Ampelisca eschrichtii Krøyer, 1842 of the Sakhalin Shelf of the Okhotsk Sea, Far Eastern Russia, comprise the highest known biomass concentration of any amphipod population in the world and are a critically important prey source for western gray whales. The high prevalence of atrophied ovaries, undersized and damaged oocytes, undersized broods of embryos and the absence of terminal phase males or females brooding fully formed juveniles among these populations in late spring and early fall are consistent with trophic stress and starvation. A. eschrichtii therefore appear to starve in summer and grow and reproduce in late fall and winter. In summer, these populations, occur below water strata containing the bulk of phytoplankton biomass and appear more likely to receive their trophic sources with vertical mixing that occurs in winter.

Ampelisca eschrichtii Krøyer, 1842 of the Sakhalin Shelf of the Okhotsk Sea, Far Eastern Russia, comprise the highest known biomass concentration of any amphipod population in the world and are a critically important prey source for western gray whales. The high prevalence of atrophied ovaries, undersized and damaged oocytes, undersized broods of embryos and the absence of terminal phase males or females brooding fully formed juveniles among these populations in late spring and early fall are consistent with trophic stress and starvation. A. eschrichtii therefore appear to starve in summer and grow and reproduce in late fall and winter. In summer, these populations, occur below water strata containing the bulk of phytoplankton biomass and appear more likely to receive their trophic sources with vertical mixing that occurs in winter.

Histological and histochemical investigation of the compound eye of the whiteleg shrimp (Litopenaeus vannamei)https://peerj.com/preprints/34532017-12-082017-12-08Hao-Kai ChangCheng-Chung LinShih-Ling Hsuan
The compound eye is the primary visual system in crustaceans. Although the histological structure and histochemical characteristics of compound eyes of some insect and crab species are now well understood, no such studies have been undertaken in the whiteleg shrimp (Litopenaeus vannamei). In this study, eye samples from L. vannamei were fixed and paraffin sections were stained using several histochemical methods. The histological structure of each layer of the compound eye was examined and compared using different histochemical staining methods. It was found that the compound eye of L. vannamei consisted of cuticle, cornea, ommatidia, optic nerve layer, lamina ganglionaris, and medulla in an outside-in order. The cuticle of L. vannamei eyes was very thin, composed of a single epicuticle layer, as confirmed by Masson’s trichrome stain. The screening pigments produced by screening pigment cells were arranged at the junction of the ommatidia and optic nerve layer; these pigments stained differentially after different histochemical staining methods suggesting the screening pigment cells can be classified into different types. Notably, clusters of foamy glandular cells (FGCs) were observed in the optic nerve layer; these stained positively with periodic acid-Schiff and toluidine blue, and appeared blue after Masson’s trichrome stain. Immunohistochemical (IHC) staining was used to further define the origin and characteristics of FGCs. The IHC analysis showed that FGCs were positive for vimentin and synaptophysin (SYN), suggesting their neuroendocrine nature. In the medulla internalis and medulla terminalis, the neural clusters that surround the neurophil could be divided into three types by differences in morphology: the largest and the smallest cell clusters were neuron clusters and neurosecretory cells, respectively; the middle-sized cell clusters appeared SYN-positive and have not previously been described. Overall, this study is the first to provide a detailed description of the normal features of the compound eye of L. vannamei. The identification of different types of screening pigments in the ommatidia, the endocrine nature of FGcs in the optic nerve layer, and the novel neural clusters between the medulla internalis and medulla terminalis, will be important information for further study into the compound eye of L. vannamei.

The compound eye is the primary visual system in crustaceans. Although the histological structure and histochemical characteristics of compound eyes of some insect and crab species are now well understood, no such studies have been undertaken in the whiteleg shrimp (Litopenaeus vannamei). In this study, eye samples from L. vannamei were fixed and paraffin sections were stained using several histochemical methods. The histological structure of each layer of the compound eye was examined and compared using different histochemical staining methods. It was found that the compound eye of L. vannamei consisted of cuticle, cornea, ommatidia, optic nerve layer, lamina ganglionaris, and medulla in an outside-in order. The cuticle of L. vannamei eyes was very thin, composed of a single epicuticle layer, as confirmed by Masson’s trichrome stain. The screening pigments produced by screening pigment cells were arranged at the junction of the ommatidia and optic nerve layer; these pigments stained differentially after different histochemical staining methods suggesting the screening pigment cells can be classified into different types. Notably, clusters of foamy glandular cells (FGCs) were observed in the optic nerve layer; these stained positively with periodic acid-Schiff and toluidine blue, and appeared blue after Masson’s trichrome stain. Immunohistochemical (IHC) staining was used to further define the origin and characteristics of FGCs. The IHC analysis showed that FGCs were positive for vimentin and synaptophysin (SYN), suggesting their neuroendocrine nature. In the medulla internalis and medulla terminalis, the neural clusters that surround the neurophil could be divided into three types by differences in morphology: the largest and the smallest cell clusters were neuron clusters and neurosecretory cells, respectively; the middle-sized cell clusters appeared SYN-positive and have not previously been described. Overall, this study is the first to provide a detailed description of the normal features of the compound eye of L. vannamei. The identification of different types of screening pigments in the ommatidia, the endocrine nature of FGcs in the optic nerve layer, and the novel neural clusters between the medulla internalis and medulla terminalis, will be important information for further study into the compound eye of L. vannamei.

Kill two birds with one stone: making multi-transgenic pre-diabetes mouse models through insulin resistance and pancreatic apoptosis pathogenesishttps://peerj.com/preprints/34372017-11-282017-11-28Siyuan KongJinxue RuanKaiyi ZhangBingjun HuYuzhu ChengYubo ZhangShulin YangKui Li
Background. Type 2 diabetes, a chronic disease to which susceptibility is hereditary, is characterized by insulin resistance accompanied by defective insulin secretion. Mouse models, especially transgenic mice, play an important role in medical research. However, the transgenic mouse models that have been used in diabetes research are involved with single transgenes, focusing on the insulin gene or its mutants. Thus they mainly provide information related to Type 1 diabetes.
Methods. Here, we attempted to focus comprehensively on genes related to pancreatic islet damage, peripheral insulin resistance and related environmental inducing factors by generating single-transgenic mice (CHOP), dual-transgenic mice (hIAPP-CHOP) and triple-transgenic mice (11β-HSD1-hIAPP-CHOP). The latter two types of transgenic animals were induced with high-fat, high-sucrose diets (HFHSD). We evaluated and analyzed the diabetes-related symptoms and the histopathological and immunohistochemical features of the transgenic animals.
Results. Specifically, in the triple-transgene animals, the results of intraperitoneal glucose tolerance tests (IPGTT) began to change 60 days after induction (p<0.001). After 190 days of induction, the body weights (p<0.01) and plasma glucose levels of the animals in the Tg group were higher than those of the animals in the Nc group. After the mice were sacrificed, large amounts of lipid were found deposited in the adipose tissues (p<0.01) and ectopically deposited in the non-adipose tissues (p<0.05 or 0.01) of the animals in the Tg HFHSD group. The weights of the kidneys and hearts of the Tg animals were significantly increased (p<0.01). Serum C-P was decreased due to transgene effects, and insulin levels were increased due to the effects of the high-fat high-sucrose diet in the Tg HFHSD group, indicating that damaged insulin secretion and insulin resistance hyperinsulinemia existed simultaneously in these animals. The serum corticosterone levels of the animals in the Tg group were slightly higher than those of the Nc animals due to the effects of the 11βHSD-1 transgene and obesity. In the Tg HFHSD group, hepatic adipose deposition was more severe and the pancreatic islet area was enlarged under compensation, accompanying apoptosis. In the Tg ControlD group, hepatic adipose deposition was also severe, pancreatic islets were damaged, and their areas were decreased (p<0.05), and apoptosis of pancreatic cells occurred. Taken together, these data show that the transgenes led to early-stage pathological changes characteristic of type 2 diabetes in the triple-transgene HFHSD group. The disease of triple-transgenic mice was more severe than that of dual or single-transgenic mice.
Conclusion. The use of multi-transgenes involved in insulin resistance and pancreatic apoptosis is a better way to generate polygene-related early-stage diabetes models.

Background. Type 2 diabetes, a chronic disease to which susceptibility is hereditary, is characterized by insulin resistance accompanied by defective insulin secretion. Mouse models, especially transgenic mice, play an important role in medical research. However, the transgenic mouse models that have been used in diabetes research are involved with single transgenes, focusing on the insulin gene or its mutants. Thus they mainly provide information related to Type 1 diabetes.

Methods. Here, we attempted to focus comprehensively on genes related to pancreatic islet damage, peripheral insulin resistance and related environmental inducing factors by generating single-transgenic mice (CHOP), dual-transgenic mice (hIAPP-CHOP) and triple-transgenic mice (11β-HSD1-hIAPP-CHOP). The latter two types of transgenic animals were induced with high-fat, high-sucrose diets (HFHSD). We evaluated and analyzed the diabetes-related symptoms and the histopathological and immunohistochemical features of the transgenic animals.

Results. Specifically, in the triple-transgene animals, the results of intraperitoneal glucose tolerance tests (IPGTT) began to change 60 days after induction (p<0.001). After 190 days of induction, the body weights (p<0.01) and plasma glucose levels of the animals in the Tg group were higher than those of the animals in the Nc group. After the mice were sacrificed, large amounts of lipid were found deposited in the adipose tissues (p<0.01) and ectopically deposited in the non-adipose tissues (p<0.05 or 0.01) of the animals in the Tg HFHSD group. The weights of the kidneys and hearts of the Tg animals were significantly increased (p<0.01). Serum C-P was decreased due to transgene effects, and insulin levels were increased due to the effects of the high-fat high-sucrose diet in the Tg HFHSD group, indicating that damaged insulin secretion and insulin resistance hyperinsulinemia existed simultaneously in these animals. The serum corticosterone levels of the animals in the Tg group were slightly higher than those of the Nc animals due to the effects of the 11βHSD-1 transgene and obesity. In the Tg HFHSD group, hepatic adipose deposition was more severe and the pancreatic islet area was enlarged under compensation, accompanying apoptosis. In the Tg ControlD group, hepatic adipose deposition was also severe, pancreatic islets were damaged, and their areas were decreased (p<0.05), and apoptosis of pancreatic cells occurred. Taken together, these data show that the transgenes led to early-stage pathological changes characteristic of type 2 diabetes in the triple-transgene HFHSD group. The disease of triple-transgenic mice was more severe than that of dual or single-transgenic mice.

Conclusion. The use of multi-transgenes involved in insulin resistance and pancreatic apoptosis is a better way to generate polygene-related early-stage diabetes models.

Short-term gains in histology knowledge: A veterinary gaming applicationhttps://peerj.com/preprints/34212017-11-202017-11-20Eric B. BaumanGregory E GilbertGreg Vaughan
Outcomes research exists incorporating non-digital games in veterinary education; however, little research examines outcomes from digital applications. Bauman’s Layered-Learning Model was applied to investigate whether a digital matching game produced short-term knowledge gains in veterinary students. Two groups of students (n1=67; n2=55) practiced the matching game for an hour. A Wilcoxon signed rank test tested for a statistically significant improvement in pre-test/post-test scores. All statistical analyses were done using R. Mean and median pre-test scores were 15 (SD=1.88; IQR: 2) and post-test scores 16 (SD=1.80; IQR: 2). Mean and median pre-test score for Group 2 were 13 (SD=2.30; IQR=2.5) and post-test scores 15 (SD=1.93; IQR=2.5). Both groups increased significantly in post-test scores (P value<.0001). Statistically significant short-term increases of marginal importance in histology knowledge was seen. A greater increase might be seen with quiz revision increasing difficulty and discrimination ability. Further directions should investigate long-term knowledge retention.

Outcomes research exists incorporating non-digital games in veterinary education; however, little research examines outcomes from digital applications.Bauman’s Layered-Learning Model was applied to investigate whether a digital matching game produced short-term knowledge gains in veterinary students. Two groups of students (n1=67; n2=55) practiced the matching game for an hour. A Wilcoxon signed rank test tested for a statistically significant improvement in pre-test/post-test scores. All statistical analyses were done using R. Mean and median pre-test scores were 15 (SD=1.88; IQR: 2) and post-test scores 16 (SD=1.80; IQR: 2). Mean and median pre-test score for Group 2 were 13 (SD=2.30; IQR=2.5) and post-test scores 15 (SD=1.93; IQR=2.5). Both groups increased significantly in post-test scores (P value<.0001). Statistically significant short-term increases of marginal importance in histology knowledge was seen. A greater increase might be seen with quiz revision increasing difficulty and discrimination ability. Further directions should investigate long-term knowledge retention.

Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent modelhttps://peerj.com/preprints/29842017-05-192017-05-19Parvathi HaridasJacqui A McGovernSean DL McElwainMatthew J Simpson
Background: Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.
Methods: 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin layers is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.
Results: Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells.
Discussion: The MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

Background: Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.

Methods: 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin layers is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.

Results: Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells.

Discussion: The MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxicationhttps://peerj.com/preprints/28782017-03-172017-03-17Anais CastagnolaJerreme JacksonOmaththage P PereraCris OppertShigetoshi EdaJuan Luis Jurat-Fuentes
Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we report the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. Biological activity of the Cry1Ac-induced secretome was monitored as higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified 4 proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin protein not detected in the control secretome. Feeding of purified arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify arylphorin as a protein with a putative relevant role in the midgut regeneration process in response to Cry1Ac intoxication.

Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we report the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. Biological activity of the Cry1Ac-induced secretome was monitored as higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified 4 proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin protein not detected in the control secretome. Feeding of purified arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify arylphorin as a protein with a putative relevant role in the midgut regeneration process in response to Cry1Ac intoxication.

Transmissible cancer in marine bivalves: Implications for mode of cancer transmissionhttps://peerj.com/preprints/28002017-02-102017-02-10Wenfa Ng
Cancer spread away from the primary tumour towards other parts of the body is what is most feared in cancer treatment, and is the leading cause of relapse for patients who are in remission after initial rounds of treatment with surgery, radiation or chemotherapy. Typically more aggressive than the primary tumour, secondary tumours developed through the process of metastasis are seeded by circulating tumour cells that migrate away from solid tumours to distant body sites. But increasingly, there is another method by which cancer is able to spread: transmission between members of the same or different species. Dangerous for the implications that it portends, the intra- and interspecies transmission of cancer highlights the potential infectious nature of the disease of neoplastic cell growth, and the implications it may have on transmission of cancer through the food chain, even though the latter link find no verifiable reported episode, but which remains a possibility to be vigilant for. Currently, transmissible cancers have been detected in dogs, Tasmanian devils, and molluscs. In a recent report in Nature, Goff and coworkers reported the widespread transmission of independent cancer lineages within and between marine bivalves species (“Widespread transmission of independent cancer lineage within multiple bivalve species”). Field studies conducted along the Pacific Northwest coast led to the identification of neoplastic bivalves, which upon sequencing of the mitochondrial cytochrome c oxidase gene proffer a set of single nucleotide polymorphisms (SNP) that could be clustered into distinct cancer lineages in a phylogenetic tree. Using sequence information as guide and histology observations as clues, clear evidence of cancer transmission between and within bivalves species were confirmed, with affected bivalves showing clear phenotypic neoplasms. Given that bivalves are a source of nutrients and vitamins in the delicacy segment of Asian cuisine, possible transmission of cancer between bivalves and humans as well as mammals such as bears that feed on them is an important direction for future research.

Cancer spread away from the primary tumour towards other parts of the body is what is most feared in cancer treatment, and is the leading cause of relapse for patients who are in remission after initial rounds of treatment with surgery, radiation or chemotherapy. Typically more aggressive than the primary tumour, secondary tumours developed through the process of metastasis are seeded by circulating tumour cells that migrate away from solid tumours to distant body sites. But increasingly, there is another method by which cancer is able to spread: transmission between members of the same or different species. Dangerous for the implications that it portends, the intra- and interspecies transmission of cancer highlights the potential infectious nature of the disease of neoplastic cell growth, and the implications it may have on transmission of cancer through the food chain, even though the latter link find no verifiable reported episode, but which remains a possibility to be vigilant for. Currently, transmissible cancers have been detected in dogs, Tasmanian devils, and molluscs. In a recent report in Nature, Goff and coworkers reported the widespread transmission of independent cancer lineages within and between marine bivalves species (“Widespread transmission of independent cancer lineage within multiple bivalve species”). Field studies conducted along the Pacific Northwest coast led to the identification of neoplastic bivalves, which upon sequencing of the mitochondrial cytochrome c oxidase gene proffer a set of single nucleotide polymorphisms (SNP) that could be clustered into distinct cancer lineages in a phylogenetic tree. Using sequence information as guide and histology observations as clues, clear evidence of cancer transmission between and within bivalves species were confirmed, with affected bivalves showing clear phenotypic neoplasms. Given that bivalves are a source of nutrients and vitamins in the delicacy segment of Asian cuisine, possible transmission of cancer between bivalves and humans as well as mammals such as bears that feed on them is an important direction for future research.